US8040513B2 - Dual emission microscope - Google Patents
Dual emission microscope Download PDFInfo
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- US8040513B2 US8040513B2 US12/141,378 US14137808A US8040513B2 US 8040513 B2 US8040513 B2 US 8040513B2 US 14137808 A US14137808 A US 14137808A US 8040513 B2 US8040513 B2 US 8040513B2
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- 230000009977 dual effect Effects 0.000 title description 2
- 230000003595 spectral effect Effects 0.000 claims abstract description 29
- 238000003384 imaging method Methods 0.000 claims abstract description 9
- 230000003287 optical effect Effects 0.000 claims description 20
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 230000004075 alteration Effects 0.000 description 7
- 238000000926 separation method Methods 0.000 description 5
- 230000001419 dependent effect Effects 0.000 description 3
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/0092—Polarisation microscopes
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B17/00—Systems with reflecting surfaces, with or without refracting elements
- G02B17/02—Catoptric systems, e.g. image erecting and reversing system
- G02B17/026—Catoptric systems, e.g. image erecting and reversing system having static image erecting or reversing properties only
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens
Definitions
- the present invention relates to a microscope device, which is capable of producing images of a sample in different spectral ranges (“colors”) or polarisations on a single detector.
- FIG. 1 A schematic of the W-view design as used in the prior art is shown in FIG. 1 .
- a collimated image beam 10 originating from an intermediate image (which is not shown in FIG. 1 ) impinges onto a first dichroic beam splitter 12 and is separated into a first beam 14 and a second beam 16 of different color, i.e. the first beam 14 essentially consists of light of a first spectral range and the second beam 16 essentially consists of light of a second spectral range.
- the first beam 14 which is reflected by the first beam splitter 12 , passes to a mirror 18 from where it is directed onto a second dichroic beam splitter 20 , which has the same spectral characteristics as the first beam splitter 12 .
- the second beam 16 which is transmitted by the first beam splitter 12 , is reflected by a second mirror 22 and directed onto the second beam splitter 20 , where the first beam 14 and the second beam 16 are “reunited” (or “combined”) in their general direction.
- the two beams 21 corresponding to the first beam 14 , and 27 , corresponding to the second beam 16 , exhibit a slight angular offset relative to each other, i.e. they diverge relative to each other to a certain degree, so as to yield the desired spatial separation on the detector chip (not shown in FIG. 1 ).
- color separation takes place in an infinity space of the optical beam path, which could be the space between the objective lens and the tube lens.
- a suitable field-stop has to reduce the field of view seen by the camera of the detector to one half of its original size, in order to accommodate the two semi-images projected side by side. Having an intermediate image requires a second set of optics (relay-lenses), which create another infinity space where beam separation takes place.
- One major advantage of this design is that using a beam splitter not only for separating beams, but also for reuniting them, allows maintaining telecentric optics throughout, thus avoiding vignetting and asymmetrical light-cones which are different for different areas on the detector chip. Moreover, the fact that both beam paths are transmitted by the same optics warrants that both color channels experience identical magnification and need no resealing before being compared. This is particularly important in co-localization studies.
- a single dichroic beam splitter may be used for separating a collimated image beam originating from an intermediate image into two different color channels, which are imaged by a common lens onto a common detector chip in order to obtain spatially separated semi-images on the detector.
- Each of the color channels is reflected twice prior to being projected onto the detector, whereas according to the prior art system shown in FIG. 1 , both color channels are reflected 3 times or, in the original Kinosita paper, one color channel is not reflected at all whereas the other one is reflected 4 times
- JP 2004361391 A wherein splitting of the two color channels and double-reflection in each channel occurs in the space between the projection lens and the detector. All prior art has in common that the number of reflections for the two beam-paths are such that both color channels have the same handedness on the chip.
- this object is achieved by a microscope device as defined in claims 1 and 16 , respectively, which enables separate “color channels” and by a microscope device as defined in claims 25 and 26 , respectively, which enables separate “polarisation channels”.
- the invention is beneficial in that, by providing the means for reflecting the one beam in a manner so as to invert its handedness and the means for reflecting the second beam in a manner so as to preserve its handedness, a fully symmetrical configuration is obtained, where corresponding image points in both color/polarisation channels all experience the same field-dependent aberrations, whereas in the prior art systems, which either maintain the handedness of both color/polarisation channels or invert handedness of both color/polarisation channels, image points, which are close to the center line in one image are close to the edge of the image in the other image, so that their aberrations differ.
- FIGS. 2 a and 2 b This is schematically illustrated in FIGS. 2 a and 2 b , where the symmetry of the images resulting from the two color channels on the detector with regard to the symmetry of the multi-color intermediate image is shown for the prior art concepts ( FIG. 2 a ) and for the present invention ( FIG. 2 a ).
- each color/polarisation channel undergoes at least one additional reflection after having been separated from the other color/polarisation channel. This concept enables comfortable adjustment of the position of each of the color/polarisation channels on the camera-chip.
- the means for reflecting the second beam and the separating means are integrated within a single member.
- FIG. 1 is a schematic view of a color splitting arrangement for a microscope device having the “W-view design” according to the prior art
- FIGS. 2 a and 2 b are schematic views of the image symmetry obtained by the color splitting arrangement of FIG. 1 of the prior art and a color splitting arrangement according to the present invention
- FIG. 3 a is a schematic view of a microscope device according to the invention comprising a first embodiment of a color splitting arrangement
- FIG. 3 b is a schematic view of the arrangement of FIG. 3 a seen in the direction of the arrow A of FIG. 3 a;
- FIG. 4 a is a schematic view of a microscope device according to the invention comprising a second embodiment of a color splitting arrangement
- FIG. 4 b is a schematic view of the arrangement of FIG. 4 a seen in the direction of the arrow A;
- FIG. 4 c is a schematic view of the arrangement of FIG. 4 a seen in the direction of the arrow B;
- FIG. 5 a is a view like FIG. 4 a , with a third embodiment of a color splitting arrangement of a microscope device according to the invention being shown;
- FIGS. 5 b to 5 d are a view of the arrangement of FIG. 5 a seen in the direction of the arrow A, wherein the incident multi-color image beam, the outgoing first beam/color channel and the outgoing second beam/color channel, respectively, are shown.
- FIGS. 3 a and 3 b show a first embodiment of a microscope device according to the invention, wherein a collimated multi-color beam 10 is generated by collecting light from a sample 11 by a compound microscope 13 (in the drawing consisting of objective 13 a and tube lens 13 b ).
- the microscope creates an intermediate image 15 located in the focal plane of a projection lens 17 .
- the light collected from the sample 11 will be emission light, in particular fluorescence emission light, such as emission light obtained from Fluorescence Resonance Energy Transfer (FRET).
- FRET Fluorescence Resonance Energy Transfer
- a first dichroic beamsplitter 12 serves to reflect one wavelength component of the beam 10 (if the dichroic is a long-pass, beam 14 is the short-wavelength-part of beam 10 ) toward a mirror 18 , thereby generating a first beam 14 , whereas the component of the beam 10 , which is transmitted by dichroic 12 , constitutes a second beam 16 , which is directed towards a roof prism 23 . While the first beam 14 is reflected by the mirror 18 towards a second dichroic beamsplitter 20 , having the same spectral characteristics as the first beam splitter 12 , the second beam 16 is deflected by the roof prism 23 in such way that it meets the first beam 14 at the second beam splitter 20 .
- the former is transmitted and the latter is reflected, thus reuniting beams 14 and 16 into a “combined” beam-bundle, consisting of beam 21 (formerly beam 14 ) and beam 27 , formerly beam 16 (the reunited beams 21 and 27 are “combined” in the sense that they later pass through the same optical elements).
- the roof-prism 23 must be oriented in such a manner that its ridge 25 is located within the plane defined by the first beam 14 and the second beam 16 .
- Beams 21 and 27 are projected by a projection lens 26 onto a detector 28 located in the focal plane of the lens 26 , so that an image of the sample 11 is generated on the active area of the detector 28 .
- the intermediate image 15 is confined to the boundaries of about half of the size of the active area of the detector 28 .
- FIG. 3 a showing the beam-splitting part of the microscope device in a view in the direction of the arrow A of FIG. 3 a , discloses that in the plane perpendicular to the paper plane of FIG. 3 a there is a slight angular offset between the beam portion 21 and the beam portion 27 .
- This angular offset is turned into a spatial offset of the images on the detector 28 (not shown) by means of the projecting lens.
- the exact angular offset—and hence the corresponding spatial offset— is controlled by appropriate relative adjustment of the elements 12 , 18 , 20 and 23 .
- two images of the sample 11 in two different spectral ranges which are determined by the beam splitters 12 , 20 can be obtained side by side on a single detector 28 .
- the first beam 14 undergoes an odd number of reflections, namely three, so that the handedness of the first beam 14 is inverted with regard to the handedness of the incident multi-color beam 10 , when being projected onto the detector 28 .
- the roof prism 23 acts as a retroreflector, with the second beam 16 undergoing an even number, namely two, reflections, so that the handedness of the second beam 16 is maintained with regard to handedness of the incident multi-color beam 10 , when being projected on the detector 28 .
- the roof prism 23 acts as a retro-reflector only in one dimension, namely with regard to the direction perpendicular to the paper plane of FIG. 3 a , whereas within the paper plane of FIG. 3 a it acts as a “normal” reflector in that the incident angle equals the outgoing angle of the beam.
- FIG. 2 b shows the handedness of the intermediate image 15 and the handedness of the resulting images 30 and 32 obtained on the detector 28 by projection of the outgoing portion 21 of the first beam 14 and the outgoing portion 27 of the second beam 16 , respectively. It can be seen that portions of the intermediate image 15 located close to the axial center line will be also located close to the axial center line for both final images, so that a fully symmetrical configuration is achieved wherein corresponding image points all experience the same field dependent aberrations when being projected by the projection lens 26 .
- FIGS. 4 a through 4 c show a modified embodiment wherein only a single dichroic beamsplitter 112 is used, instead of two dichroic beamsplitters 12 and 20 as in FIGS. 3 a , 3 b .
- only a single projection lens 126 is used. Its purpose is not only to collimate beam 110 , which originates from the intermediate image 115 , but also to project the outgoing beams 37 and 38 next to each other onto the detector 128 .
- the intermediate image 115 of the sample 111 is located in the focal plane of the projection lens 126 and is confined to the boundaries of about half of the active area of the detector 128 . It is off-center relative to the optical axis 140 of the projection lens 126 in both the dimension displayed in FIG. 4 a (this off-center position allows separating the intermediate image 115 from the image on the detector 128 ) and in the dimension shown in FIG. 4 b , which is perpendicular to the plane displayed in FIG. 4 a.
- the beam 110 Before the beam 110 intersects the optical axis 140 in the focal plane of the lens 126 , it reaches a dichroic beamsplitter 112 which serves to separate the incident beam 110 into a first beam 114 which is transmitted by the beamsplitter 112 onto a mirror 118 and a second beam 116 , which is reflected by the beam splitter onto a roof prism 23 .
- the mirror 18 is located in the focal plane of the lens 126 and serves to reflect the first beam 114 back to the beamsplitter 12 , where it is transmitted again.
- the mirror 118 is adjusted in such a manner that the out-going portion 121 of the first beam 114 has an angular offset with regard to the incident beam 110 in the paper plane of FIG. 4 a .
- the roof prism 123 is arranged in such a manner that the ridge 125 of the prism 123 is located in the paper plane of FIG. 4 a (in the shown example, the ridge 125 is parallel to the central optical axis 140 ) and that the second beam 116 received from the beamsplitter 12 is reflected back to the beamsplitter 12 , where it is reflected again in such a manner that it forms an outgoing portion 127 which coincides in the view of FIG. 4 a with the outgoing portion 121 of the first beam 114 reflected by the mirror 118 and transmitted by the beam splitter 112 in order to form a combined beam 124 .
- the prism 123 acts as a retro-reflector, whereas it acts as a “normal” reflector in the dimension in the paper plane of FIG. 4 a.
- the reflecting elements 112 , 118 and 123 are adjusted in such a manner that the outgoing portions 121 and 127 of the first beam 114 and the second beam 116 have a slight angular offset relative to each other in the direction perpendicular to the paper plane of FIG. 4 a , so that the image of the first beam 114 and the image of the second beam 116 on the detector 128 have a spatial offset.
- the outgoing portions 121 and 127 of the first beam 114 and the second beam 116 pass through the projection lens 126 and, now called beam 37 and 38 , form separate images in the focal plane of lens 126 .
- a prism 142 may be provided in the outgoing beams 37 and 38 for deflecting these beams onto the detector 128 , thus facilitating their separation from the incoming beam 110 .
- the prism 142 may also be placed in the incoming beam 110 .
- FIGS. 5 a to 5 d show an embodiment which is a simplified version of that of FIGS. 4 a to 4 c in that the functions of the beam splitter 112 , the mirror 118 and the roof prism 123 are integrated into a single element 223 .
- the intermediate microscope image 215 is radially shifted with regard to the optical axis 240 of the projection lens 226 not only in the dimension extending in the paper plane of FIG. 5 a , but also in the dimension extending perpendicular to the paper plane of FIG. 5 a , see FIG. 5 b .
- the intermediate image 215 is located in the focal plane of the projection lens 226 .
- the collimated multi-color beam 210 is angled towards the optical axis 240 and impinges onto the element 223 , which is a roof prism having a dichroic coating 218 on the front surface.
- the ridge 225 of the roof prism 223 is arranged in the paper plane of FIG. 5 a , and the coated front surface 218 is essentially perpendicular to the optical axis 240 .
- the dichroic surface 218 serves to split the incoming beam 210 into an outgoing first beam 221 , which is reflected at the surface 218 towards the projection lens 226 , and a second beam 216 , which is transmitted by the surface 218 into the interior of the roof prism 223 , where it is reflected back to the surface 218 .
- the roof prism 223 acts as a retro-reflector in the dimension perpendicular to the paper plane of FIG. 5 a , whereas it acts as a “normal” reflector in the dimension extending in the paper plane of FIG. 5 a
- the collimated incident beam 210 is angled towards the optical axis 240 also with regard to that dimension (see FIG. 5 b ). Since with regard to that dimension the roof prism 223 acts as a retro-reflector, the second beam 216 transmitted by the surface 218 is reflected back in the direction of the incoming multi-color beam 210 , so that—apart from the displacement of the combined beam 224 with regard to the incident multi-color beam 210 in the paper plane of FIG.
- beams 221 and 227 exhibit the same angle relative to the optical axis 240 , but having opposite signs. This warrants that the images created from the two beams 221 and 227 by the projecting lens 226 are next to each other in the plane of the detector 228 , in FIGS. 5 b and 5 c above and below the optical axis 240 but both touching it. Given that the first outgoing beam 221 has experienced one reflection in both dimensions whereas the outgoing portion 227 of the second beam 216 undergoes one reflection in one and two reflections in the other dimension, their respective images created by the projection lens 226 differ in their handedness.
- the dichroic beamsplitter could be either a short pass or a long pass.
- the above embodiments which serve to provide for two separate color channels, also could be used to realize two separate polarisation channels.
- the light collected from the sample would include two different polarisations and the dichroic beamsplitters would be replaced by beamsplitters which split the incoming mixed polarisation beam into two beams having different polarisation.
Abstract
Description
Claims (26)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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US12/141,378 US8040513B2 (en) | 2008-06-18 | 2008-06-18 | Dual emission microscope |
US13/275,109 US8427646B2 (en) | 2008-06-18 | 2011-10-17 | Dual emission microscope |
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US12/141,378 US8040513B2 (en) | 2008-06-18 | 2008-06-18 | Dual emission microscope |
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US13/275,109 Continuation US8427646B2 (en) | 2008-06-18 | 2011-10-17 | Dual emission microscope |
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US13/275,109 Active US8427646B2 (en) | 2008-06-18 | 2011-10-17 | Dual emission microscope |
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Cited By (2)
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US8427646B2 (en) | 2008-06-18 | 2013-04-23 | Fei Company | Dual emission microscope |
US20140160265A1 (en) * | 2011-04-14 | 2014-06-12 | Fei Company | Switchable microscope arrangement with multiple detectors |
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CN103162831B (en) * | 2011-12-19 | 2014-12-10 | 中国科学院微电子研究所 | Broadband polarization spectrograph and optical measurement system |
DE102012205722B4 (en) * | 2012-04-05 | 2020-07-23 | Carl Zeiss Microscopy Gmbh | Imaging color splitter module, microscope with such a color splitter module and method for imaging an object field in a first and a second image plane |
JP6285112B2 (en) * | 2013-06-03 | 2018-02-28 | 浜松ホトニクス株式会社 | Light splitter |
GB201318919D0 (en) | 2013-10-25 | 2013-12-11 | Isis Innovation | Compact microscope |
CN103698008B (en) * | 2013-12-24 | 2015-09-16 | 安徽三兴检测有限公司 | A kind of spectrometer for industrial field |
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GB201507021D0 (en) | 2015-04-24 | 2015-06-10 | Isis Innovation | Compact microscope |
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JPWO2021161684A1 (en) * | 2020-02-13 | 2021-08-19 |
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US8040513B2 (en) | 2008-06-18 | 2011-10-18 | Till I.D. Gmbh | Dual emission microscope |
-
2008
- 2008-06-18 US US12/141,378 patent/US8040513B2/en active Active
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- 2011-10-17 US US13/275,109 patent/US8427646B2/en active Active
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US5793523A (en) * | 1995-07-14 | 1998-08-11 | J.D. Moller Optische Werke Gmbh | Beam divider more particularly for optical instruments such as operating microscopes |
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US8427646B2 (en) | 2008-06-18 | 2013-04-23 | Fei Company | Dual emission microscope |
US20140160265A1 (en) * | 2011-04-14 | 2014-06-12 | Fei Company | Switchable microscope arrangement with multiple detectors |
US9759901B2 (en) * | 2011-04-14 | 2017-09-12 | Fei Company | Switchable microscope arrangement with multiple detectors |
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US20090316258A1 (en) | 2009-12-24 |
US20120176488A1 (en) | 2012-07-12 |
US8427646B2 (en) | 2013-04-23 |
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